The principal goal of this project is to develop recombinant HIV-1 vaccine constructs that elicit both humoral and cellular immunity in both the mucosal and systemic compartments after oral immunization. To this end, we will construct fusions between gp120, or gp120 fragments, and the A (LTA) and B (LTB) chains of Escherichia coli. heat labile enterotoxin (LT) as mucosal targeting systems. LT binds to GM1 gangliosides on mucosal cell surfaces via its 5 LTB subunits (LTB5) and mediates toxicity by delivery of its single LTA subunit to the cytoplasm where it is responsible for ADP- ribosylation of GSalpha leading to adenylate cyclase activation. LTA has two domains, A1 and A2, that are responsible for toxicity and association with LTB5, respectively. We will develop non-toxic chimeras between gp120 and the non-toxic A2 domain of LTA (LTA-A2) or the C-terminus of LTB that retain the ability to bind GM1 gangliosides and that will deliver gp120 epitopes to the immune system via the mucosal route. Both purified chimeras and the chimeras expressed in attenuated Salmonella vaccine strains will be evaluated for the abilities to induce humoral and cellular immunity in the mucosal and systemic compartments. There are 4 specific aims. Aim 1: To systematically develop LTB::gp120 chimeras that bind GM1 gangliosides and express gp120 epitopes. Component 1: Determination of optimal spacer length and composition between LTB and a T1::V3 epitope pair. Component 2: Determination of the maximum sized fragment of gp120 that can be expressed as an LTB::spacer::g120 chimera. Component 3: Each chimera will be evaluated initially for in vitro stimulation of Class I and Class II restricted gp120 specific T cells using different antigen presenting cells. This information will be used to decide which chimeras to express in attenuated Salmonella typhimurium in Aim 3 and which chimeras to evaluate in vivo as purified proteins in Aim 4. Aim 2: To systematically develop LTA-A2::gp120 chimeras that assemble with wild-type LTB to form LTB5:LTA-A2::gp120 complexes. Component 1: Determination of optimal spacer length and composition between LTA and a T1::V3 epitope pair. Component 2: Development of dual-epitope LTA::gp120 chimeras that express two copies of a T1::V3 epitope pair. Component 3: Determination of the maximum sized fragment of gp120 that can be expressed as an LTA::spacer:g120 chimera. Component 4: Each chimera will be evaluated initially for in vitro stimulation of class I and class II restricted gp120 specific T cells using different antigen presenting cells. This information will be used to decide which chimeras to express in attenuated Salmonella typhimurium in Aim 3 and which chimeras to evaluate in vivo as purified proteins in Aim 4. Aim 3: To express selected LTB::gp120 chimeras in attenuated Salmonella carrier strains. Aim 4: To perform preclinical immunogenicity trials in mice for selected chimeras developed in the above Aims.